单叶省藤三种组织RNA提取和全长cDNA文库构建  

Comparison of Three RNA Isolation Methods for Three Tissues of Calamus simplicifolius and Construction of a Full-length cDNA Library

作　　者：杨帆[1,2] 李发根[1] 翁启杰[1] 李梅[1] 洪亚辉[2] 甘四明[1] 

机构地区：[1]中国林业科学研究院热带林业研究所,广州510520 [2]湖南农业大学生物科学技术学院,长沙410128

出　　处：《分子植物育种》2010年第4期800-803,共4页Molecular Plant Breeding

基　　金：国家科技支撑计划(2006BAD19B0901);国家高技术研究发展计划重点项目(2006AA100109)共同资助

摘　　要：本实验比较了3种RNA提取方法提取单叶省藤的茎、雄花序和根的结果,改良CTAB法和Trizol法提取的RNA无明显降解、完整性好,均可用于cDNA文库构建,而异硫氰酸胍法提取的RNA降解严重;考虑到改良CTAB法成本较低,故推荐为首选方法。将茎、雄花序和根三种组织的RNA等量混合后,利用SMART策略构建了全长cDNA文库;通过涂平板培养后的菌落计数,测得初库滴度为2.91×105cfu/μL;从培养平板上随机挑取16个菌落进行PCR检测,文库重组率为87.5%,插入片段平均长度为1.5kb左右。所建文库的质量可靠,为下一步的EST测序和候选基因发现提供了材料基础。In this research,by comparing the results among three procedures for isolating total RNA from stem,male inflorescence and root of Calamus simplicifolius,we found that modified CTAB method and Trizol method were better than guanidnium isothiocyanate method on yield and quality of RNA from all the three tisues.As modified CTAB method was more cost-effective than the Trizol,it was recommended as the sound choice of RNA isolation.Then,we constructed a full-length cDNA library in accordance with SMART strategy and by using a mixture of RNA of the three tissues.The titer of the library was 2.91×10^5 cfu/μL.PCR of 16 colonies selected randomly from the culture indicated that the recombination rate and mean insert size were 87.5% and around 1.5 kb,respectively.The cDNA library constructed here should be qualified for subsequent EST sequencing and candidate gene discovery in C.simplicifolius.

关 键 词：单叶省藤 RNA提取 CDNA文库 

分 类 号：S564.1[农业科学—作物学]